P1366: SAR442257, A CD38/CD28/CD3 TRISPECIFIC ANTIBODY, POTENTIATES CAR T-CELL ACTIVITY AGAINST LARGE B-CELL LYMPHOMA
Authors: Patrick Reville, Enyu Dai, Irtiza Sheikh, Qing Deng, Jared Henderson, Cuong Le, Estela Rojas, Chizitara Okwuchi, Ashley Wilson, Sydney Parsons, Shyanne Lockman, Peter Kim, Kamlesh Bisht, Helgi van de Velde, Hongfang Wang, Sattva Neelapu, Linghua Wang, Michael Green
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: CAR T cell therapy has significantly improved outcomes of patients with relapsed/refractory large B cell lymphoma (rrLBCL), but many do not experience long term benefit. The mechanisms of failure are multifaceted and includes CAR T-cell exhaustion. Understanding these mechanisms are likely to identify rational therapeutic avenues for improving outcomes. Aims: 1.To understand the impact of the rrLBCL immune microenvironment on clinical failure to CAR T therapy 2.Investigate targeting of CD38 with a novel CD38 T cell engaging trispecific antibody in combination with CD19 CAR T-cells Methods:Discovery analysis: 13 rrLBCL tumors, including 7 CAR T naïve and 6 CAR T refractory tumors, were subjected to scRNA-seq. Clustering was performed and frequency, clonal dominance, and expression profiles of major cell types and subclusters were compared between groups. Functional experiments: The effect of SAR442257 was assessed in cytotoxicity assays and compared to control antibodies lacking the CD38 or CD38/CD3/CD28 targeting regions. RL and HT lymphoma cell lines were CRISPR modified to knock-out CD19 and isogenic WT clones used as target cells. CD19 CAR T-cells were constructed from PBMCs of rrLBCL patients obtained at the time of apheresis using a construct like axicabtagene ciloleucel (axi-cel). For cytotoxicity assays, cell lines were co-cultured at E:T ratios of 1:1 for 24h or 48h. Results: scRNA-seq revealed that CAR T refractory rrLBCL tumors possessed significantly higher fractions of terminally exhausted LAG3+TIM3+CD38+ CD8 T-cells (CD8TEX) with high expression of T-cell dysfunction and TEX signatures compared to CAR T naïve tumors. CD8TEX were most frequent in CAR T refractory tumors and enriched within CAR+CD8 T-cells detected within all CAR T refractory tumors. We have previously identified similar cells enriched in axi-cel infusion products of rrLBCL patients failing to response (Deng et al., Nat. Med. 2020). TCR clonotype analysis revealed highly expanded T-cell clones within the CD8TEX cluster, significantly increased clonal dominance, and reduced clonal diversity of CD8TEX cells in CAR T refractory compared to CAR T naïve tumors. Single cell differential gene expression revealed significantly increased expression of LAG3, TIM3, and CD38 in CD8 T-cells from CAR T refractory compared to CAR T naïve tumors. We hypothesized that SAR442257, a CD38/CD28xCD3 trispecific antibody, could boost the activity of CD19 CAR T-cells from rrLBCL patients in part by allowing for dual antigen targeting of CD19 and CD38 on lymphoma cells and by inducing fratricide of CD38-expressing CD8TEX cells. Cytotoxicity assays showed that SAR442257 significantly increased the killing of CAR T-cells against HT and RL WT (CD19+/CD38+) cells (24-hr HT P-value=3e-4, 24-hr RL P-value=1e-2) which persisted at 48 hours. Addition of SAR442257 to CAR T-cells allowed killing of HT and RL CD19KO (CD19-/CD38+) (24-hr HT P-value=8e-7, 24-hr RL P-value=2e-8) at a level similar to the combination in WT cells. Addition of antibodies lacking CD38 or CD38/CD3/CD28-targeting regions did not boost CAR T cytotoxicity or rescue killing of CD19KO targets. We observed significant T-cell fratricide, which was beneficial or non-detrimental in light of significantly increased lymphoma cell killing. Summary/Conclusion: The tumor microenvironment of CAR T refractory rrLBCL is enriched in clonally expanded and terminally exhausted CD8 T-cells expressing CD38. The CD38/CD28xCD3 trispecific antibody SAR442257 boosted CAR T-cell activity through recognition of CD38 on the tumor, costimulation of CAR T-cells, and induced fratricide of CD38+ T-cells; resulting in superior tumor cell killing. In addition, SAR442257 allowed CD19 CAR T-cells to kill CD19-/CD38+ LBCL cells. Keywords: Diffuse large B cell lymphoma, Cellular therapy, Lymphoma therapy, CAR-T